RESEARCH ARTICLE Open Access

A unique thymus-derived regulatory T cellsubset associated with systemic lupuserythematosusHironari Hanaoka1†, Tetsuya Nishimoto1†ˆ, Yuka Okazaki2, Tsutomu Takeuchi1 and Masataka Kuwana1,2*

Abstract

Background: Foxp3 is a marker for regulatory T cells (Treg cells), but recent studies have shown the plasticity andheterogeneity of CD4+Foxp3+ T cells. This study aimed to examine the phenotype and function of circulatingCD4+Foxp3+ T cells in patients with systemic lupus erythematosus (SLE).

Methods: We enrolled 47 patients with SLE, 31 with organ-specific autoimmune diseases (15 with multiple sclerosisand 16 with primary immune thrombocytopenia), and 19 healthy subjects. Peripheral blood mononuclear cells wereused to evaluate the proportion and phenotype of CD4+Foxp3+ cells using multicolor flow cytometry, the status ofthe Treg-specific demethylated region (TSDR) of the foxp3 gene by methylation-specific polymerase chain reaction,and the immunoregulatory function of CD4+CD25+ cells by allogeneic mixed lymphocyte reaction.Immunohistochemistry of renal biopsy specimens obtained from 6 patients with lupus nephritis and 5 with IgAnephropathy was conducted to detect IL-17A-expressing CD4+Foxp3+ cells.

Results: CD4+Foxp3+ T cells were increased in SLE patients compared with organ-specific autoimmune diseasecontrols or healthy controls. Circulating CD4+Foxp3+ T cells were correlated with the disease activity of SLE. Theincreased CD4+Foxp3+ T cells in active SLE patients were mainly derived from thymus-derived Treg (tTreg) cells, asdetermined by a demethylated TSDR status, and represented a unique phenotype, upregulated expression ofCD49d, CD161, and IL-17A, with immunosuppressive ability comparable to that of healthy controls. Finally,CD4+Foxp3+IL-17A+ cells were infiltrated into the renal biopsy specimens of patients with active lupus nephritis.

Conclusions: A unique tTreg subset with dichotomic immunoregulatory and T helper 17 phenotypes is increasedin the circulation of SLE patients and may be involved in the pathogenic process of SLE.

Keywords: Systemic lupus erythematosus, Regulatory T cells, Th17, T cell plasticity

© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you giveappropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtainpermission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to thedata made available in this article, unless otherwise stated in a credit line to the data.

* Correspondence: kuwanam@nms.ac.jpˆTetsuya Nishimoto is deceased.1Division of Rheumatology, Department of Internal Medicine, Keio UniversitySchool of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan2Department of Allergy and Rheumatology, Nippon Medical School GraduateSchool of Medicine, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan

Hanaoka et al. Arthritis Research & Therapy (2020) 22:88 https://doi.org/10.1186/s13075-020-02183-2

BackgroundForkhead box P3 (Foxp3)-positive regulatory T cells(Treg cells) are key mediators of peripheral self-tolerance that are able to actively suppress effector Tcells, inhibit inflammation, and prevent autoimmunity[1, 2]. The expression of Foxp3, considered a masterregulator of Treg cell development and function, is es-sential for the role of Treg cells in the maintenance ofimmune tolerance, but recent studies have shown plasti-city and heterogeneity within CD4+Foxp3+ T cells, whichinclude thymus-derived Treg (tTreg) cells, which are dir-ectly developed from CD4 single-positive cells in thethymus, peripherally derived Treg (pTreg) cells, whichare differentiated from naïve CD4+ T cells in the periph-ery, and a subpopulation of activated effector T cells [3,4]. Functionality of tTreg cells strongly depends on ex-pression of Foxp3, but tTreg cells are not a homoge-neous cell population and show diverse functionalproperties with various expression of surface markersand soluble mediators [5].It is known that numeric abnormalities and functional

impairment of Treg cells potentially contribute to thepathogenesis of various autoimmune diseases, includingrheumatoid arthritis (RA) and organ-specific auto-immune diseases such as myasthenia gravis (MG), im-mune thrombocytopenia (ITP), and multiple sclerosis(MS) [6–11]. Furthermore, genetic mutations in thefoxp3 gene lead to functional impairment of Treg cells,resulting in the development of severe autoimmune andinflammatory conditions [12].Systemic lupus erythematosus (SLE) is characterized

by a breakdown of peripheral tolerance to a variety ofself-antigens, followed by activation and expansion ofautoreactive effector T and B cells, resulting in multipleorgan damage through production of pathogenic auto-antibodies and resultant immune complex deposition[13]. It has been shown that dysregulated adaptive andinnate immune systems contribute to the pathophysi-ology of SLE [14, 15]. Since Treg cells play a major rolein maintaining immune tolerance in the periphery, thenumbers and function of CD4+Foxp3+ T cells in SLE pa-tients have been extensively studied in recent years [16–23]. However, these studies have demonstrated quitecontradictory results: some reported a reducedfrequency and/or impaired regulatory function of circu-lating Foxp3+ Treg cells [19–21] in SLE patients in com-parison to healthy controls, but others found anincreased or comparable frequency of circulating Foxp3+

Treg cells [22, 23]. A recent meta-analysis revealed thatthe pooled percentage of CD4+Foxp3+ T cells in activeSLE patients was found to be lower than that in controls,with great heterogeneity [24]. These discrepancies likelyarise from the heterogeneity of CD4+Foxp3+ T cells andthe difference in the combination of markers used in the

flow cytometric analysis. Nevertheless, in this study, weinvestigated CD4+Foxp3+ T cell subsets associated withSLE by focusing on the heterogeneity of phenotypes andfunction of CD4+Foxp3+ T cells.

MethodsPatients and controlsThis study used peripheral blood samples from 47patients with SLE, who were consecutive patients visitinga rheumatology clinic at Keio University Hospital. Allpatients fulfilled the 1997 American College of Rheuma-tology revised criteria for the classification of SLE [25].Patients taking > 20 mg of a prednisolone equivalentdaily were excluded. Nineteen age/sex-matched healthysubjects were used as a control. In addition, 15 patientswith MS and 16 with primary ITP were used as diseasecontrols, since MS and ITP were shown to have dysregu-lated Treg/Th17 balance that potentially contributes tothe pathogenesis [11]. All patients with MS or primaryITP satisfied the published criteria [26, 27].We also used renal biopsy specimens of patients with

lupus nephritis, independent of the analysis using per-ipheral blood samples. A reason for selecting kidneysamples for the analysis was simply the availability of theaffected organ samples obtained from SLE patients.Renal biopsy specimens were randomly selected fromour renal biopsy bank: 6 samples of diffuse proliferativelupus nephritis classified as class IV-G (A/C) accordingto the International Society of Nephrology/Renal Path-ology Society classification [28] and 5 samples of histo-logically confirmed IgA nephropathy. All samples wereobtained after the subjects gave their written informedconsent, as approved by the Institutional Review Board.

Clinical characteristicsThrough a retrospective chart review conducted at thesame time as blood sampling or renal biopsy, demo-graphic and clinical features, laboratory results, andtreatment regimens were recorded in individual SLE pa-tients. We also recorded the SLE disease activity index(SLEDAI) [29], 50% complement hemolytic activity(CH50) value, and the titers of serum anti-double-stranded DNA (dsDNA) antibodies, which were mea-sured using a commercial enzyme-linked immunosorb-ent assay kit (MESACUP® DNA-II test, MBL, Nagoya,Japan) according to the manufacturer’s instructions.Active SLE was defined as not satisfying Lupus Low Dis-ease Activity State [30], and mild, moderate, and severedisease activity were defined according to the recom-mendations [31].

Cell preparationPeripheral blood mononuclear cells (PBMCs) were iso-lated from heparinized venous blood using Lymphoprep

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(Fresenius Kabi AG, Bad Homburg, Germany) densitygradient centrifugation. In some experiments, CD4+

cells, CD4+CD25+ cells, and CD4+CD25− cells were iso-lated from PBMCs using magnetic cell sorting (MACS)column separation (Miltenyi Biotec, Bergisch Gladbach,Germany) according to the manufacturer’s protocol. Inaddition, a FACS Vantage flow cytometer (Becton Dick-inson, Franklin Lakes, NJ, USA) was used to isolateCD4+Foxp3+ cells and CD4+Foxp3− cells from PBMCs.The sorted fraction consistently contained > 90% of tar-geted cells as confirmed by flow cytometric analysis.

Flow cytometryFor multicolor flow cytometric analysis, PBMCs werestained with the following fluorescence-conjugated mono-clonal antibodies against CD4 (clone 13B8.2; BeckmanCoulter Inc., Indianapolis, IN, USA), CD25 (clone M-A251; Becton Dickinson), CD49d (clone 9F10; BectonDickinson), CD127 (clone HIL-7R-M21; Becton Dickin-son), CD152 (clone L3D10; Biolegend, San Diego, CA,USA), and CD161 (clone HP-3G10; Biolegend). Cells werethen fixed and permeabilized using the anti-human Foxp3staining set (eBioscience, San Diego, SC, USA) followed byintracellular staining with monoclonal antibodies againstFoxp3 (clone 236A/E7; eBioscience), IFN-γ (clone B27;Biolegend), IL-2 (clone MQ1–17H12; Biolegend), IL-17(clone N49–653; Biolegend), and Helios (clone 22F6; Bio-legend) according to the manufacturer’s instructions. Thecells were analyzed on a FACS Calibur (Becton Dickinson)or FACS MoFlo (Beckman Coulter Inc.). In some experi-ments, the expression level of Foxp3 was evaluated usingthe mean fluorescent intensity (MFI).

Analysis of the methylation status of the Treg-specificdemethylated region (TSDR)Demethylation of the TSDR within the promoter regionof the foxp3 gene is associated with stable expression ofFoxp3 and is a unique property of tTreg cells [32, 33].To evaluate the methylation status of the TSDR,methylation-specific polymerase chain reaction (PCR)was used as previously described [34]. Briefly, genomicDNA was extracted from isolated CD4+Foxp3+ andCD4+Foxp3− cells with the QIAamp DNA blood minikit (Qiagen Inc., Valencia, CA, USA) and then subjectedto bisulfite conversion with the Cell-to-CpG™ BisulfiteConversion kit (Applied Biosystems, Foster City, CA,USA) according to the manufacturer’s instructions.Bisulfite-treated genomic DNA was subjected to real-time PCR to analyze the methylation status of the TSDRusing methylated DNA or demethylated DNA-specificprobes and primers [34]. The proportion of cells with ademethylated TSDR was calculated using the followingformula: [number of demethylated TSDR sequences] /[sum of number of demethylated and methylated TSDR

sequences] × [number of X chromosomes per cell]. Allanalyses were conducted in duplicate.

Analysis of immunosuppressive functionThe immunosuppressive capacity of CD4+CD25+ T cellswas examined using allogeneic mixed lymphocyte reac-tion (MLR) as previously described [35]. Briefly,CD4+CD25− cells (104), which were used as effector Tcells, were labeled with 1 μM carboxyfluorescein succini-midyl ester (CFSE) (Invitrogen) and cultured with irradi-ated (60 Gy) allogeneic PBMCs (105) as feeder cells inRPMI 1640 medium supplemented with 10% fetal bovineserum, 100 IU/mL penicillin, and 100 mg/mL strepto-mycin in the presence or absence of CD4+CD25+ T cells(104), which were used as Treg cells. Next, 96-wellround-bottom plates were used after pretreatment with0.5 μg/mL anti-CD3 monoclonal antibody (clone OKT3;Becton Dickinson). Phytohemagglutinin (PHA) was usedas a control for T cell proliferation. After 5 days of cul-ture, the proliferation of CFSE-labeled cells was assessedby flow cytometry. Immunosuppressive activity was cal-culated based on the following formula: (1-[proportionof proliferating cells in the culture of effector T cells andTreg cells] / [proportion of proliferating cells in the cul-ture of effector T cells]) × 100 (%).

ImmunohistochemistryInfiltration of IL-17-expressing CD4+Foxp3+ T cells wasevaluated by immunohistochemistry of renal biopsyspecimens as previously described with some modifica-tions [34]. Briefly, paraffin-embedded sections (5-μmthickness) of renal biopsy samples were boiled in 10mMcitrate buffer (pH 6.0) and blocked with 5% bovineserum albumin. Subsequently, the sections were incu-bated with anti-CD4 monoclonal antibody (1:100, clonemAbcam51312, Abcam), anti-Foxp3 monoclonal anti-body (1:50, clone mAbcam22510, Abcam), and anti-IL-17A rabbit polyclonal antibody (1:100, Abcam), followedby incubation with fluorescence-conjugated secondaryantibodies (Molecular Probes). Images were taken usinga Fluoview FV1000 confocal laser fluorescence micro-scope (Olympus, Tokyo, Japan). The proportions ofFoxp3+ cells in CD4+IL-17A+ cells were determined bycounting at least 50 cells of the cells positive for IL-17A.

Statistical analysisContinuous data are shown as box-whisker plots or themean ± standard deviation. The box plots contain a cen-tral rectangle (box) with lines (whiskers) that extendfrom both ends and provide information about the smal-lest value, first quartile (Q1), median, third quartile (Q3),and largest value. All statistical analyses were performedusing IBM SPSS statistics 22 (IBM Corporation,Armonk, NY, USA). Comparisons between two groups

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were tested for statistical significance using the nonpara-metric Mann-Whitney U test. Comparisons among 3 ormore groups were performed using ANOVA followed byBonferroni’s test. The correlation coefficient was ob-tained by Spearman’s correlation analysis. P < 0.05 wasconsidered significant.

ResultsDemographic and clinical characteristics of SLE patientsused for peripheral blood analysisThe age at enrollment was 42.4 ± 14.8, 44.9 ± 9.1, 51.5 ±16.7, and 42.2 ± 10.8 years, and the proportion of femaleswas 70%, 60%, 50%, and 68% in patients with SLE, MS,primary ITP, and healthy controls, respectively. Therewas no difference in age and sex distribution amonggroups, while age tended to be older in patients with pri-mary ITP than that in the other groups. The clinicalcharacteristics of the SLE patients used for analysis areshown in Table 1. The disease activity, distribution oforgan involvement, and treatment were variable amongpatients: this was expected because consecutive patientsin routine clinical practice were enrolled. Nevertheless, itis apparent that the enrolled subjects included thepatients with apparent disease activity with elevatedanti-dsDNA antibody levels and increased SLEDAI atblood draw. Lupus nephritis was manifested in 42.5% of

the patients. SLE patients took a mean of 10.9 ± 9.6 mg/day of prednisolone with a variety of immunosuppressiveagents. No patients took hydroxychloroquine.

Increased proportion of CD4+Foxp3+ T cells in thecirculation of active SLE patientsWe first examined the frequencies of Foxp3+ cells in cir-culating CD4+ T cells using flow cytometry (Fig. 1). Theproportion of CD4+Foxp3+ T cells was increased in SLEpatients compared to healthy controls, patients with MS,or those with primary ITP (P < 0.01 in all comparisons).In SLE patients, the proportion of CD4+Foxp3+ T cellswas positively correlated with anti-dsDNA antibody titer(r = 0.57, P < 0.01) and SLEDAI (r = 0.52, P < 0.01) andnegatively correlated with CH50 levels (r = 0.52, P < 0.01).Peripheral blood samples of 6 patients with moderate

or high disease activity at enrollment (4 with activenephritis with histologic confirmation of class IV-G, and2 with pleuritis, skin rash, and cytopenia) were availableafter the remission induction treatment. The treatmentregimens used for all patients were high-dose corticoste-roids combined with an immunosuppressant (4 withintravenous cyclophosphamide, and 2 with tacrolimus).In 6 SLE patients, the proportion of CD4+Foxp3+ T cellswas significantly decreased after immunosuppressivetreatment, which resulted in a reduction in SLEDAI(P < 0.01) (Fig. 2). These results together suggest thatthe increased proportion of CD4+Foxp3+ T cells incirculation is specific to SLE and correlates with diseaseactivity. Therefore, samples obtained from patients withactive SLE were used in the following experiments.

Origin of CD4+Foxp3+ T cells increased in the circulationof active SLE patientsTo assess whether the increased CD4+Foxp3+ T cells inthe circulation of SLE patients were derived from thethymus, the methylation status at the TSDR of the foxp3gene was examined by methylation-specific PCR. Toexamine whether the TSDRs of CD4+Foxp3+ T cells inSLE patients were demethylated, CD4+Foxp3+ andCD4+Foxp3− cells were individually isolated from thePBMCs of 6 active SLE patients and 6 heathy controlsusing flow cytometer-based sorting and subjected tomethylation-specific PCR (Fig. 3a and b). The TSDR ofthe foxp3 gene of CD4+Foxp3+ T cells was completelydemethylated in both SLE patients and healthy controls.This finding indicates that the increased CD4+Foxp3+ Tcells in the circulation of active SLE patients originatedmainly from tTreg cells.

Altered phenotype of CD4+Foxp3+ T cells in thecirculation of active SLE patientsWe next evaluated the phenotypes of CD4+Foxp3+ Tcells increased in circulation of active SLE patients using

Table 1 Characteristics of SLE patients used for analysis

Number 47

Age, year 42.4 ± 14.8

Female, number (%) 33 (70)

SLEDAI 5.0 ± 4.8

CH50, U/mL 31.7 ± 12.0

Anti-dsDNA antibody, U/mL 105.0 ± 134.2

CRP, mg/dL 0.4 ± 0.9

Organ manifestations

Skin, number (%) 11 (23.4)

Arthritis, number (%) 10 (21.3)

Cytopenia, number (%) 18 (38.3)

Lupus nephritis, number (%) 20 (42.5)

Serositis, number (%) 7 (14.9)

Immunosuppressive therapy

Prednisolone, mg/day 10.9 ± 9.6

Azathioprine, number (%) 10 (21.3)

Methotrexate, number (%) 9 (19.1)

Tacrolimus, number (%) 12 (25.6)

Cyclosporine, number (%) 4 (8.5)

Hydroxychloroquine, number (%) 0 (0)

SLE systemic lupus erythematosus, SLEDAI systemic lupus erythematosusdisease activity index; dsDNA, double-stranded DNA, CH50 50% complementhemolytic activity

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multicolor flow cytometry. We examined the expressionof CD25, CD45RA, CD127, CD49d, CD161, CD152, andHelios as well as cytokines, including IL-2, IL-17, andIFN-γ, on gated CD4+Foxp3+ T cells in active SLE pa-tients and healthy controls (Fig. 4). In CD4+Foxp3+ Tcells from SLE patients, CD25high cells were less frequentbut CD49d+ cells were more frequent than the respect-ive abundances in CD4+Foxp3+ T cells from heathy con-trols (P < 0.01 for both comparisons). There was nodifference in the proportions of CD45RA+, CD127+,CD152+, and Helios+ cells between SLE and healthy con-trols, whereas the proportion of CD161+ cells was mark-edly increased in active SLE patients versus healthycontrols (P < 0.01). Finally, the expression level of Foxp3within CD4+Foxp3+ T cells was significantly lower in ac-tive SLE patients than in healthy controls (P < 0.01). Interms of cytokine expression profiles, lower IL-2 andhigher IL-17A were observed in the CD4+Foxp3+ T cells

from active SLE patients compared with the respectivelevels in those from healthy controls (P < 0.05 for bothcomparisons), whereas IFN-γ expression was compar-able (Fig. 5). These findings together suggest that the in-creased CD4+Foxp3+ T cells in the circulation of activeSLE patients consist mainly of Foxp3lowCD45RA+C-D25low cells with higher expression of CD49d, CD161,and IL-17A but comparable expression of CD152 andHelios.

Immunosuppressive function of CD4+CD25+ T cells in thecirculation of active SLE patientsThe in vitro immunosuppressive potency of CD4+CD25+

T cells was assessed using cultures of allogeneic MLR(Fig. 6). For this purpose, we isolated CD4+CD25+ andCD4+CD25− cells from PBMCs of active SLE patientsand healthy controls as Treg cells and effector T cells,respectively. The isolated CD4+CD25+ cells constantly

Fig. 1 Proportion of circulating CD4+Foxp3+ T cells in SLE patients and controls. a Representative dot plot analysis in a healthy donor and a patientwith SLE. b Proportion of CD4+Foxp3+ T cells in healthy controls (n = 19), SLE patients (n = 47), MS patients (n = 15) and primary ITP patients (n = 16).c Correlations between the proportion of CD4+Foxp3+ T cells and anti-double-stranded DNA (dsDNA) antibody levels, CH50 values, and SLE diseaseactivity index (SLEDAI) scores in 47 patients with SLE

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contained > 90% CD4+Foxp3+ T cells. In representativeassays using samples from an active SLE patient and ahealthy control, the proliferation of effector T cells in-duced by allogeneic PBMCs was similarly suppressed bycoculture with CD4+CD25+ cells. There was no differ-ence in the immunosuppressive activity of CD4+CD25+

T cells between active SLE patients and healthy controls.

Foxp3 expression in CD4+IL-17A+ cells infiltrated intorenal biopsy specimensWe further examined the potential recruitment ofCD4+Foxp3+IL-17A+ T cells in the affected organs of

SLE patients. For this purpose, renal biopsy specimensfrom patients with active lupus nephritis and those withIgA nephropathy were randomly selected from our renalbiopsy bank, and subjected to immunohistochemistry(Fig. 7). Clinical characteristics of the 6 patients withclass IV-G (A/C) lupus nephritis at renal biopsy includedproteinuria of 4.7 ± 0.7 g/day, glomerular filtration rateof 82.4 ± 0.7 mL/min/1.73 m2, SLEDAI of 14.3 ± 1.9,anti-dsDNA antibody levels of 148.7 ± 28.9 U/mL, andCH50 of 18.7 ± 0.9 U/mL, on treatment with 13.0 ± 4.7mg/day of prednisolone without immunosuppressant.CD4+IL-17A+ cells were detected mainly in the intersti-tial lesions of kidneys from both patients with lupus

Fig. 2 Serial analysis of the proportion of circulating CD4+Foxp3+ T cells in patients with active SLE. a Representative dot plot analysis in a patientwith SLE before and after immunosuppressive treatment. b Changes in SLEDAI score and the proportion of CD4+Foxp3+ T cells before and aftertreatment in 6 patients with SLE

Fig. 3 Methylation status at the TSDR of the foxp3 gene in CD4+Foxp3+ T cells in active SLE patients and healthy controls. a A representative dotplot of the target cell populations sorted using a FACS Vantage flow cytometer: CD4+Foxp3+ cells and CD4+Foxp3− cells. b The mean proportionof demethylated TSDRs in isolated CD4+Foxp3+ cells and CD4+Foxp3− cells in healthy controls (n = 6) and active SLE patients (n = 6)

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nephritis and those with IgA nephropathy, but the ma-jority of the CD4+IL-17A+ cells also showed nuclear/cytoplasmic expression of Foxp3 in patients with lupusnephritis. The proportion of Foxp3+ cells in CD4+IL-17A+ cells was significantly greater in patients withlupus than in those with IgA nephropathy. Correlationsof proportions of CD4+Foxp3+IL-17A+ T cells in thekidney and peripheral blood were not evaluable because

peripheral blood samples of the patients at renal biopsywere not available.

DiscussionOur results demonstrated that circulating CD4+Foxp3+

T cells were increased in SLE patients in associationwith disease activity. Most of the increased CD4+Foxp3+

T cells in the peripheral blood of active SLE patients

Fig. 4 Phenotypic properties of CD4+Foxp3+ T cell subsets in active SLE patients and healthy controls. a Representative dot-plot analysis in ahealthy donor and a patient with active SLE. b Proportions of the cells with Treg and Th17 markers, including CD25high, CD45RA, CD49d, CD127,CD152, Helios, and CD161, in CD4+Foxp3+ T cells from healthy controls (n = 15) and active SLE patients (n = 16). c The expression level of Foxp3in CD4+Foxp3+ T cells from healthy controls (n = 15) and active SLE patients (n = 16). NS = not significant

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originated from tTreg cells, as determined by the com-pletely demethylated status at the TSDR of the foxp3gene, and were consistent with naïve or resting Tregcells, as reported by Miyara et al. [36]. This tTreg subsetrepresented a unique phenotype, upregulated expressionof CD49d, expression of the authentic Treg markersHelios and CD152, expression of the T helper 17 (Th17)marker CD161, and expression of IL-17A, and the cellsubset showed immunosuppressive ability. Finally, IL-17A-expressing CD4+Foxp3+ T cells were also detectedin renal biopsy specimens of patients with active lupusnephritis. Since depressed Treg cell function and ele-vated Th17 response are critical to the pathogenesis ofSLE [37], these results together indicated that a uniquetTreg subset acquiring both immunoregulatory andTh17 phenotypes might be involved in the pathogenicprocess of SLE by expanding in circulation and infiltrat-ing into the affected tissue. It is interesting to note thatthis unique tTreg subset has two different aspects interms of SLE pathogenesis, including immunosuppres-sive and proinflammatory properties, although our study

failed to draw any conclusion whether they are harmfulor protective.Our findings were principally consistent with previous

studies showing increased tTreg cells with a demethylatedTSDR of the foxp3 gene locus in the peripheral blood ofSLE patients [36, 38–41]. Although the markers used toevaluate CD4+Foxp3+ T cell subsets were variable amongprevious studies, resulting in difficulty in comparing thefindings described in individual reports, the CD4+Foxp3+

T cell subsets reported to be increased in SLE patients in-cluded Foxp3lowCD45RA+CD25low cells (fraction I) [36],Helios+ T cells [40, 41], CD161+ T cells [42], and IL-17-producing T cells [39], which were consistent with theunique tTreg subset identified in this study.It has been recently reported that Treg cells become

unstable under certain inflammatory and/or pathologicconditions and adopt characteristics of effector CD4+ Tcells [3, 11, 43]. In particular, Foxp3+ Treg cells are ableto differentiate into IL-17-producing Th17-like cellsupon receiving external signals. The first study in miceshowed that transforming growth factor-β induced

Fig. 5 Cytokine expression profiles of CD4+Foxp3+ T cells in active SLE patients and healthy controls. a Representative dot plot analysis for theexpression of IFN-γ and IL-17 on CD4+Foxp3+ T cells from a healthy donor and a patient with active SLE. b Proportions of T cells expressing IL-2,IL-17, IFN-γ, or both IFN-γ and IL-17 in CD4+Foxp3+ T cells from healthy controls (n = 11) and active SLE patients (n = 11). NS = not significant

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differentiation of CD4+Foxp3+RORγt+ T cells with theability to produce IL-17 [44], but subsequent studiesusing human peripheral blood and secondary lymphoidtissue identified similar CD4+Foxp3+ T cell subsets pro-ducing IL-17 and inhibiting the proliferation of CD4+ re-sponder T cells [42, 45]. These CD4+Foxp3+ T cellsubsets with both Treg and effector T cell propertieswere characterized by a demethylated status of TSDRsof the foxp gene locus, immunosuppressive function, andproduction of pro-inflammatory cytokines, including IL-17 [46]. There is accumulating evidence showing theplasticity of Treg cells and their role in autoimmunity,

and these bipotential Treg cell subsets have been shownto be involved in pathogenic processes in a variety ofautoimmune mouse models, such as arthritis, nephritis,and colitis [43, 47, 48]. In addition, CD4+ T cells ex-pressing both Foxp3 and IL-17 have been shown to beincreased in the peripheral blood of patients with variousautoimmune diseases, including systemic sclerosis, RA,and Crohn’s disease [35, 47, 49, 50], although theanalysis of TSDRs of the foxp3 gene locus was not con-ducted in some studies, resulting in difficulty in discrim-inating between CD4+Foxp3+ T cell subsets originatingfrom tTreg cells, pTreg cells, and effector T cells.

Fig. 6 In vitro immunosuppressive potency of CD4+CD25+ T cells assessed by allogeneic mixed lymphocyte reaction in active SLE patients andhealthy controls. Allogeneic peripheral blood mononuclear cells (allo-PBMCs) were cocultured with flow cytometer-sorted CD4+CD25+ cells,which were regarded as T regulatory cells (Treg cells), and/or CD4+CD25− cells, which were regarded as effector T cells (Teff cells). Cellproliferation was quantitatively assessed using CSFE labeling. PHA was used as a control for cell proliferation. a Representative histograms for cellproliferation in a healthy donor and a patient with SLE. b Immunosuppressive activity of CD4+CD25+ T cells in active SLE patients (n = 5) andhealthy controls (n = 5). NS = not significant

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Nevertheless, Komatsu et al. [47] reported infiltration ofCD4+Foxp3+IL-17A+ T cells in the inflamed synoviumof RA patients, similar to our findings observed in therenal tissue of SLE patients, suggesting the pathologicalimportance of plastic tTreg cells in systemic auto-immune diseases. We used renal biopsy specimens asone of the affected organs of SLE due to sample avail-ability, it is interesting to examine whether CD4+Fox-p3+IL-17A+ T cells are recruited to other manifestedorgans of SLE.One of the notable features of IL-17-expressing tTreg

cells detected in active SLE patients is high expressionof CD49d, also called α4 integrin, which forms a com-plex with β1 or β7 integrin. There is little information

on the function of CD49d+ Treg cells, but one study re-ported a lower immunosuppressive capacity ofCD4+CD45RA− effector T cells than CD49d− Treg cells[51]. CD49d is involved in the migration of activated ef-fector CD4+ T cells, including Th1 cells and Th17 cells,into inflamed tissue, such as the central nervous sys-tem, intestine, and kidneys [45, 52, 53]. In a rat auto-immune experimental nephritis model, reduction in theCD49d expression on lymphocytes and the resultant in-hibition of their adhesion led to complete resolution ofnephritis [54]. Therefore, it is possible that CD49dexpressed on Th17-like tTreg cells contributes to pref-erential infiltration of potential pathogenic T cells intoaffected tissue.

Fig. 7 Detection of infiltrating CD4+Foxp3+IL-17+ T cells in renal biopsy samples from patients with lupus nephritis and IgA nephropathy. a Representativeimages of immunohistochemical staining for CD4 (cell surface), IL-17A (cytoplasm), and Foxp3 (cytoplasm and nucleus). Magnification=× 400. b Proportions ofFoxp3+ T cells in CD4+IL-17+ T cells infiltrated in renal specimens from patients with lupus nephritis (n = 6) and those with IgA nephropathy (n = 5)

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It has been shown that the fate of tTreg cells towardsdifferentiation into inflammatory T cells is mediated in asignal transducer and activator of transcription 3(STAT3)-dependent manner [55, 56]. In this regard,Th17-like cells can be induced from tTreg cells in vitroby stimulation with a combination of high-mobilitygroup box-1 protein (HMGB-1) and IL-6 through en-hanced STAT3 signaling [39]. A critical role of IL-6 ininducing Th17 cells has been well recognized [57].HMGB-1 is a damage-associated molecular pattern(DAMP) and a ligand of Toll-like receptor (TLR)2 andTLR4 [58]. It is interesting to note that HMGB-1 is re-leased from apoptotic cells, which are abundant in thecirculation and lymphoid tissues of patients with activeSLE [59]. On the other hand, IL-21 stimulated mamma-lian target of rapamycin (mTOR) complexes 1 and 2, ab-rogated the autophagy, differentiation, and function ofTreg cells in a STAT3-dependent manner, and drove ex-pansion of Th17-like cells in SLE patients [60, 61]. Up-regulation of IL-21 was also reported in patients withactive SLE [62]. Finally, it is known that IL-2 regulatesthe homeostasis of CD4+ T cells, and its deficiency maylead to the instability of Treg cells in patients with SLE[63, 64]. Further investigations of intracellular mecha-nisms regulating differentiation of tTreg cells into in-flammatory T cells in SLE patients are necessary toprovide insights into pathogenesis of immune dysregula-tion observed in SLE patients.In mice, IL-2 enhances Treg cell development and sur-

vival and suppresses the differentiation of follicularhelper T cells and Th17 subsets [65]. A recent studyevaluating the efficacy of low-dose IL-2 treatment in pa-tients with SLE found an increase in the number of Tregcells and suppression of follicular helper T cells andTh17 cell numbers in peripheral blood, accompanied bya marked reduction in disease activity [66]. A recent in-triguing report described the first case of a patient withSLE treated with autologous adoptive Treg cell therapy,which led to increased activated Treg cells in the in-flamed skin, with a marked attenuation of the IFN-γpathway and a reciprocal augmentation of the IL-17pathway [67]. This phenomenon was more pronouncedin skin than in peripheral blood and was validated in amouse model undergoing Treg cell adoptive transfer.These findings suggest that dysregulation of immuneand inflammatory systems in SLE patients plays a role inconverting tTreg cells into Th17-like cells.

ConclusionsWe have demonstrated that a dichotomic tTreg subsetwith both immunoregulatory and Th17 phenotypes is in-creased in the circulation of SLE patients. This tTregsubset might be involved in the pathogenic process ofSLE by infiltrating into the effected tissue, although

whether this subset is harmful or protective remains un-clear. Further studies investigating the roles of this tTregsubset in the pathogenic process of SLE and the mecha-nisms underlying its differentiation are useful for under-standing the pathogenesis of SLE and developingpotential biomarkers and therapeutic targets.

AbbreviationsFoxp3: Forkhead box P3; Treg cells: regulatory T cells; tTreg: thymus-derivedTreg; pTreg: peripherally derived Treg; MG: myasthenia gravis; ITP: immunethrombocytopenia; MS: multiple sclerosis; SLE: systemic lupus erythematosus;SLEDAI: SLE disease activity index; CH50: 50% complement hemolytic activity;PBMCs: peripheral blood mononuclear cells; MACS: magnetic-activated cellsorting; TSDR: Treg-specific demethylated region; MLR: mixed lymphocytereaction; CFSE: carboxyfluorescein succinimidyl ester;PHA: phytohemagglutinin; Th17: T helper 17; STAT3: signal transducer andactivator of transcription 3; HMGB-1: high-mobility group box-1 protein;DAMPs: damage-associated molecular patterns; TLR: Toll-like receptor;mTOR: mammalian target of rapamycin

AcknowledgementsNone.

Authors’ contributionsT.N. performed the majority of research and analyzed and interpreted thedata; Y.O., H.H., and T.T. performed the research and collected samples; M.K.designed the research, interpreted the data and supervised and organizedthe study; all authors wrote the manuscript. All authors read and approvedthe final manuscript.

Authors’ informationT.N. was deceased.

FundingThis work is supported by a research grant on intractable diseases from theJapanese Ministry of Health, Labor and Welfare.

Availability of data and materialsThe datasets analyzed during the current study are available from thecorresponding author upon reasonable request.

Ethics approval and consent to participateThis study was approved by the Ethics Committee of Keio University Schoolof Medicine. We obtained informed written consent from all subjects prior tocollecting samples in accordance with the tenets of the Declaration ofHelsinki.

Consent for publicationNot applicable.

Competing interestsNone.

Received: 27 January 2020 Accepted: 7 April 2020

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